The broad, long-term objective of the work proposed herein is to validate a novel strategy for anti-cancer therapy. A hallmark of cancerous cells is their ability to resist apoptosis and become immortal. This resistance is typically due to mutations in or aberrant expression of a wide variety of proteins in the apoptotic cascade. These alterations are effectively "breaks" in the apoptotic circuitry, which prevent proapoptotic signals from being transmitted to activate procaspase-3 to caspase-3. Caspase-3 is the major "executioner" caspase that is responsible for the proteolysis of hundreds of cellular substrates. Interestingly, procaspase-3 levels are elevated in a variety of cancers, but the defective apoptotic machinery simply cannot activate this zymogen. Described herein is an anti-cancer strategy to directly activate, with a small molecule, procaspase-3 to active caspase-3. In exciting preliminary results we have, through high-throughput screening, identified a procaspase-3 activating compound that we call PAC-1. In cell culture, PAC-1 induces apoptotic cell death, and its potency is directly proportional to the amount of procaspase-3 present in the cell. PAC-1 also powerfully induced apoptotic death in cells from primary colon tumors, with a potency strictly dependent on the amount of procaspase-3 in the cells. We have shown PAC-1 to be active in three different mouse models of cancer. In the proposed work we will use this discovery of PAC-1 as a springboard to probe the apoptotic pathways, evaluate the pharmacokinetics, efficacy, and toxicity of PAC-1 and derivatives in mice, and elucidate the biochemical mechanism by which PAC-1 activates procaspase-3. Public health relevance. Cancer has now overtaken heart disease as the leading cause of death in the U.S and is an enormous public health problem. Mutations in the fundamental cell death machinery enable cancerous cells to resist natural defenses and chemotherapeutic treatments. We propose a strategy by which we will completely bypass the mutated circuitry and activate the death pathway in cancer cells, thus killing the cancer and saving the life of the patient.